JOURNAL OF BACTERIOLOGY, Mar. 1970, p. 813-820 Vol. 101, No. 3Copyright 0 1970 American Society for Microbiology Printted in U.S.A.

Physiological Comparison of L-Serine Dehydrataseand Tryptophanase from Bacillus alvei'

S. K. GRIFFITHS AND R. D. DEMOSS

Departmenit oJ Microbiology, Uniiversity of lllintois, Urbanaci, Illiniois 61801

Received for publication 13 June 1969

Tryptophanase from Bacillus alvei also possesses serine dehydratase activity. Acomparison of this enzyme with L-serine dehydratase [L-serine hydro-lyase (deami-nating), EC 4.2.1.13] in toluene-treated whole cell preparations of the organism wasundertaken. Tryptophanase is a constitutive enzyme in B. alvei. The dehydrataseundergoes a repression-derepression-repression sequence as the L-serine level in thegrowth medium is increased from 0 to 0.1 M. Tryptophanase activity is decreased inorganisms grown in medium containing glucose. Both enzymes are repressed in or-ganisms grown in glycerol-containing medium. L-Serine dehydratase has a pH opti-mum of 7.5 in potassium phosphate buffer; tryptophanase functions optimally inthis buffer at pH 8.2. Both enzymes lose activity in the presence of tris(hydroxy-methyl)aminomethane buffer. Either K+ or NH4+ is required for full tryptophanaseactivity, but Na+ is markedly inhibitory. These three cations are stimulatory to L-serine dehydratase activity. Both enzymes are subject to apparent substrate inhibi-tion at high concentrations of their respective amino acids, but the inhibition oftryptophanase activity can be completely overcome by the removal of indole as it isformed. The dehydratase does not catalyze cleavage of D-serine, L-threonine, ora-substituted serine analogues at the concentrations tested. However, activity of theenzyme in cleaving L-serine is competitively inhibited by D-serine, indicating that theD-isomer can occupy an active site on the enzyme. The enzyme catalyzes cleavage ofsome ,3-substituted serine analogues.

Tryptophanases from Escherichia coli (13)and from Bacillus alvei (S. R. O'Neil, Ph.D.Thesis, Univ. of Illinois, Urbana, 1969) havebeen shown to catalyze both a, 1-eliminationreactions and 13-replacement reactions. The majoractivity of the enzyme, that of degrading trypto-phan to indole, pyruvate, and ammonia, is ana,,B-elimination reaction. The deamination ofserine to pyruvate and ammonia is also an a ,13-elimination reaction, but, in the case of tryp-tophanase from B. alvei, this reaction is catalyzedonly 15% as efficiently as the tryptophan deg-radation reaction. Since B. alvei possesses anactive L-serine dehydratase [L-serine hydro-lyase(deaminating), EC 4.2.1.13] which catalyzesthe same serine-deaminating reaction as tryp-tophanase, it is of interest to ask why the organismpossesses two enzymes which can perform thesame function. It is also of interest to determinewhether the genes specifying both enzymesmight have arisen from a common ancestral

' A preliminary report of this work was presented at the 69thAnnual Meeting of the American Society for Microbiology,Miami Beach, Fla., 4-9 May 1969.

gene. The initial approach to these questionshas been the comparison of physiological prop-erties of the two enzymes in toluene-treatedwhole cell preparations of B. alvei. The resultsof this investigation are reported here.

MATERIALS AND METHODS

Bacterial strain. A nonmucoid colony variant ofB. alvei (ATCC 6348), isolated by- Hoch (8) anddesignated strain F, was used in the experimentsreported here.

Growth media and culture conditions. For mostexperiments, the bacteria were grown overnight in2% (w/v) Trypticase (BBL) broth which had beensupplemented with thiamine hydrochloride (10 Ag/ml)and adjusted to pH 7.1 before autoclaving.

Cells used in catabolite repression experimentswere transferred from the supplemented Trypticasemedium to the minimal salts medium of Vogel andBonner (16) supplemented with thiamine hydro-chloride (10 Ag/ml) and 1% vitamin-free, salt-freecasein hydrolysate (Nutritional Biochemicals Corp.,Cleveland, Ohio). During the exponential growthphase, the cells were transferred to fresh supple-mented minimal salts medium without further addi-

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tions (control) or to which had been added either1% glucose or 1% glycerol.

For studying the inducibility of enzymes, cellswere transferred from supplemented Trypticasemedium to the minimal salts medium supplementedthis time with thiamine hydrochloride (10,g/ml) andone of the following: 1% glucose alone, 1% glucoseand a single amino acid (20,g/ml), or a mixture of21 L-amino acids (each 20 ,ug/ml) excluding L-serine.The single amino acids were glycine, L-threonine,L-serine, and L-homoserine. The mixture of 21 aminoacids included glycine, L-alanine, L-valine, L-leucine,L-isoleucine, L-threonine, L-homoserine, L-methio-nine, L-tryptophan, L-cystine, L-glutamic acid, L-glutamine, L-aspartic acid, L-asparagine, L-arginine,L-histidine, L-phenylalanine, L-lysine, L-tyrosine,L-cysteine, and L-proline. During the exponentialgrowth phase, the cells were transferred to freshsupplemented minimal salts medium to which hadbeen added glucose, glucose and the single aminoacid, or the amino acid mixture plus various concen-trations of L-serine. Cultures were harvested at ap-proximately the same level of growth during late loggrowth phase. The specific activity of tryptophanasein B. alvei is maximal, and the specific activity ofL-serine dehydratase is still relatively high at thisstage of growth. The absorbancy reading at whichlate log growth phase occurred was dependent uponthe degree of supplementation of the culture me-dium.The ratio of culture volume to flask volume was

maintained at 1:4. All culture flasks were shaken ona New Brunswick gyratory shaker at 37 C. The ab-sorbancy of cultures was measured at 660 nm in aZeiss PMQII spectrophotometer. If the absorbancyreading was greater than 0.6, the sample was dilutedto give a more accurate turbidity measurement.Throughout the course of this investigation and underall growth conditions used, the ratio of absorbancy(turbidity) of cultures to cell protein content wasconstant. One unit of absorbancy is equivalent to0.240 mg of cell protein per ml.

Toluene-treated whole cell preparation. All prepara-tory and assay procedures were at 0 C, unless other-wise indicated. Samples of cultures were centrifugedat 10,000 X g for 10 min; the cells were washed with1 sample volume of 0.05 M potassium phosphate orother indicated buffer (pH 7.5) and suspended to anabsorbancy reading of approximately 5.0 at 660 nm.Samples (0.1 ml) of washed cells were then incubatedwith 0.05 ml of toluene at 37 C for 5 min to destroythe permeability barrier of the cells before additionof the assay mixture.

Tryptophanase assay. For most experiments, thefollowing modification of the assay procedure ofPardee and Prestidge (15) was utilized. Duplicatereaction mixtures containing, unless otherwise stated,25 ,.moles of potassium phosphate or other indicatedbuffer (pH 8.2), 16 nmoles of pyridoxal phosphate,4 MAmoles of L-tryptophan, and toluene-treated cellsuspension in a final volume of 0.5 ml were incubatedat 37 C for 0 or 10 min. Zero-time reaction mixturesprovided blank values which were later subtractedfrom the experimental (10-min) values. The reactions

were terminated by the addition of 3 ml of color re-agent (14.7 g of p-diaminobenzaldehyde, 52 ml ofconcentrated H2SO4, 948 ml of 95%, ethyl alcohol).After a 20-min color development period at roomtemperature, the tubes were centrifuged to sedimentcellular debris and the absorbancies of the super-natant fluids were measured at 568 nm in a Gilford300 spectrophotometer. Indole was used as the stand-ard.

In substrate affinity experiments, it was desirableto remove indole as it was formed. The followingmodification of the assay procedure described byBoezi and DeMoss (1) was utilized. Duplicate reac-tion mixtures containing 210 ,umoles of potassiumphosphate buffer (pH 8.2), 200 nmoles of pyridoxalphosphate, and toluene-treated cell suspension in afinal volume of 1.5 ml were overlaid with 4 ml oftoluene in 50-ml Erlenmeyer flasks and shaken slowlyat 37 C for 5 min. The reactions were initiated by theaddition of 0.5 ml of various concentrations of tryp-tophan, and the incubation was continued at 37 C withshaking for 10 min. The reactions were terminated bythe addition of 0.2 ml of 2.5 N NaOH, and the shak-ing was continued for 5 min. Samples (1.0 ml) of thetoluene layers were assayed for indole by adding 9 mlof color reagent (5.4 g of p-diaminobenzaldehyde, 64ml of concentrated H2SO4, 908 ml of 95%jC ethyl alco-hol), incubating at room temperature for 20 min, andmeasuring color intensities at 568 nm.

L-Serine dehydratase assay. Duplicate reaction mix-tures containing, unless otherwise stated, 55 sAmolesof potassium phosphate or other indicated buffer(pH 7.5), 40 nmoles of pyridoxal phosphate, 100/Amoles of L-serine, and toluene-treated cell suspensionin a final volume of 1.0 ml were incubated at 37 C for0 or 10 min. The reactions were terminated by theaddition of 1.0 ml of 10',j trichloroacetic acid. Pre-cipitated protein was removed by centrifugation. Thesupernatant fluids were assayed for keto acid (pyru-vic, fl-phenylpyruvic, or a-ketobutyric) by the directmethod of Friedemann and Haugen (3), and theresultant color intensities were measured at 515 nmin a Gilford 300 spectrophotometer. Crystallinesodium pyruvate, sodium 3-phenylpyruvate, anda-ketobutyric acid were used as standards.

Determination of intracellular serine and pyruvateconcentrations. Cultures (150 ml), grown as for in-ducibility experiments in minimal salts medium sup-plemented with thiamine hydrochloride (10 jAg/ml),the mixture of 21 amino acids (each 20 Ag/ml), andvarious concentrations of L-serine, were harvested atapproximately the same growth level during late loggrowth phase. Cultures were centrifuged at 10,000 Xg for 10 min; the cells were washed with 1 culturevolume of 0.05 M potassium phosphate buffer (pH7.5) and suspended to an absorbancy reading of ap-proximately 20.0 at 660 nm (volume of approxi-mately 5.0 ml). Suspensions were incubated with 0.1ml of toluene at 37 C for 5 min and then centrifugedat 35,000 X g for 10 min. Assay of 0.5-ml samples ofsupernatant fluids for pyruvate was as describedabove. Samples (2.5 ml) of supernatant fluids weredeproteinized by the addition of 0.5 ml of 30c% tri-chloroacetic acid; after standing for 10 min in an ice

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bath, samples were centrifuged at 35,000 X g for 10min. Resultant supernatant fluids were freed from tri-chloroacetic acid by five successive extractions, eachwith 2 volumes of ether. Residual ether was removedby evaporation into an airstream directed over thesample surface. Samples were diluted with an equalvolume of sodium citrate buffer (0.4 M with respect tosodium and 0.2 M with respect to citrate), pH 2.2;1.0-ml sample volumes were analyzed for serine con-

tent on the 150-cm column of a Beckman/Spincomodel 120 amino acid analyzer. The instrument wascalibrated with Beckman amino acid calibration mix-ture (type 1).

Protein determination. After overnight digestion of0.05-ml samples of washed whole cell suspensions inthe presence of 0.1 ml of 2.5 N NaOH at room tem-perature, protein content was determined by themethod of Lowry et al. (11). Crystalline bovine serum

albumin, similarly treated with NaOH overnight, wasused as the standard.Enzyme units and specific activity. One enzyme unit

is defined as the amount of enzyme producing 1.0jsmole of product (keto acid or indole) per min at37 C. Specific activity is expressed as units of enzymeper milligram of protein.

RESULTS AND DISCUSSIONEffect of substrate on enzyme level. In contrast

to the tryptophanase of E. coli which is inducibleat high levels of tryptophan (7, 12), tryptophanaseof B. alvei is a constitutive enzyme, not influencedby the exogenous level of tryptophan or relatedcompounds (8). In experiments reported here,tryptophanase was repressed in B. alvei cellsgrown in the presence of moderate to high levelsof L-serine (Table 1). Large amounts of indolewere excreted by cells grown in the presence oflow exogenous serine levels, but indole excretiondiminished markedly as the serine concentrationwas increased. Diminished indole excretionwould be the expected consequence of repressionof the enzyme responsible for its formation.

L-Serine dehydratase has been shown to bereadily inducible in E. coli by L-leucine andglycine, but only to a small extent by L-serine(14). In the present investigation, the level ofL-serine dehydratase in B. alvei was not influencedby the absence of amino acids or by the presenceof L-serine, glycine, L-threonine, or L-homoserineat levels of 20 gg/ml (Table 2).The enzyme, however, although appearing

to be constitutive in cells grown in the absence ofL-serine and in the presence of low and highlevels of the amino acid, was derepressed at leasttwofold in the presence of intermediate levels ofL-serine. Figure 1 shows the repression-derepres-sion-repression sequence as the L-serine levelin the growth medium was increased from 0 to0.1 M. Also note that pyruvate excretion into the

TABLE 1. Effect of exogentouis serinie coneelntratiolnOil tryptophaanase synthlesis andl

indole accumulationia

Serine Grh Specific Indoleconcn rowt activity excretedb

M

0 0.91 0.0090 0.04340.0005 1.08 0.0081 0.03260.001 0.93 0.0088 0.02060.005 1.07 0.0090 0.01660.01 1.00 0.0087 I 0.00800.02 1.07 0.0047 0.00120.04 1.12 0.0040 0.00060.10 1.08 0.0040 0.0006

" Cells were grown in minimal salts mediumsupplemented with thiamine hydrochloride (10,ug/ml), a mixture of 21 L-amino acids (each 20sg/ml), and the indicated concentration of L-serine. Growth is presented as absorbancy at 660nm; one unit of absorbancy is equivalent to 0.240mg of cell protein per ml.

b Amount (M4moles) of indole excreted into me-dium per milliliter of culture at correspondinggrowth level.

TABLE 2. Effect oJ sinigle amino acidis Oil L-seriniedehydratlse synthesis"

Amino acid Growth Specificactivity

None....... .347 144Glycine.... .343 .146L-Serine..336 .149L-Threonine .. .378 .132L-Homoserine..359 139

"The data were compiled from three separateexperiments. Cells were grown in minimal saltsmedium supplemented with thiamine hydrochlo-ride (10 gg/ml), 1% glucose, and the indicatedamino acid (20 ,g/ml). Growth is presented asabsorbancy at 660 nm; one unit of absorbancy isequivalent to 0.240 mg of cell protein per ml.

medium did not commence until derepression wasmaximal.A tentative explanation for the repression-

derepression-repression phenomenon involvedspeculation concerning internal pyruvate andserine concentrations. It was suggested that, atlow serine concentrations, pyruvate is formedpredominantly by other metabolic pathways andmight serve to repress the dehydratase. Then agradual increase in serine concentration couldoverride the repression of the enzyme by pyru-vate. However, the increased dehydratase activitywould result in the formation of larger amountsof pyruvate which could again repress the enzyme.

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FiG. 1. Effect of exogenous serine concentrationon L-serine dehydratase synthesis and pyruvate ex-cretion. Cells were grown in minimal salts mediumsupplemented with thiamine hydrochloride (10,ug/ml),a mixture of 21 L-amino acids (each 20 ,ug/ml), andvarious concentrations of L-serine. All cultures wereharvested at approximately the same level of growth(late exponential growth phase). Samples were cen-trifuged. Supernatant fluids were assayed for pyruvateas described in Materials and Methods. Cells werewashed, resuspended, toluene-treated, and assayed forL-serine dehydratase activity.

On the basis of this explanation, one mightpostulate that only at a certain narrow range ofserine concentrations is the internal pyruvateto serine ratio such that the enzyme is maximallyderepressed.However, determination of the intracellular

serine and pyruvate concentrations negated theportion of the above theory explaining the secondrepression phase of the phenomenon. The datain Table 3 indicate that both the internal serinelevel and the internal pyruvate level varieddirectly with the specific activity of the enzyme.At exogenous serine concentrations varyingfrom 0.005 to 0.1 m, the ratio of internal pyruvateto internal serine remained constant. If increasedinternal pyruvate concentration had been re-sponsible for the repression of the enzyme athigh exogenous serine concentrations, then theratio of internal pyruvate to internal serine wouldhave increased at these high exogenous concen-trations.

Since the enzyme was again repressed atexogenous serine concentrations above 0.01 M,it would seem likely that, although sufficientexogenous serine was present, the transportsystem across the cell membrane was not rapidenough to supply the additional serine necessaryto both keep the enzyme derepressed and at thesame time serve as substrate for the enzyme.Thus, with the serine transport system operatingat its maximal rate, enzyme synthesis woulddecrease to a level at which it would remain

TABLE 3. Relationship of intracellular serilie alidpyruvate concentrations to L-serinie

dehydratase synthesisa

External ~~Enzyme Internal InternalExternal |Growth specific Internal [yr [Pyruva tel[Serinel activity [Serinel vPyte- /Internalat] [Serine]

O 1.05 0.091 0.41b 1 . 58b 3.850.0001 1.12 0.124 2.07 2.41 1.160.0002 1.01 0.076 0.57 1.89 3.320.0005 1.17 0.132 6.19 3.56 0.580.001 1.08 0.127 3.67 3.17 0.860.005 1.12 0.179 10.60 3.80 0.360.01 1.06 0.302 18.49 3.32 0.180.02 1.08 0.206 9.67 3.68 0.380.04 1.12 0.097 5.00 1.80 0.360.10 1.14 0.100 1.75 0.65 0.37

aGrowth conditionsTable 1.

were as described in

bExpressed as nanomoles of compound withinorganisms present in 1.0 ml of culture at indicatedgrowth level.

constant regardless of any further increase inexogenous serine concentration.

This explanation is supported by the recentwork of Kay and Gronlund (10). They demon-strated that serine is in the group of seven aminoacids exhibiting the lowest rates of transport intowhole cells of Pseudomonas aeruginosa and thatthe amino acid transport system of the organismbecomes saturated at high external amino acidconcentrations. Serine was shown to have a19-fold lower rate of transport than, for example,leucine.

Effect of catabolites on enzyme synthesis andproduct excretion. It had been reported previously(J. A. Hoch, Ph.D. Thesis, Univ. of Illinois,Urbana, 1965) that tryptophanase from B. alveiis not subject to catabolite repression. The currentinvestigation has shown that the enzyme isdefinitely subject to repression by glucose and toan even greater extent by glycerol (Fig. 2). Al-though during the middle period of exponentialgrowth (4th to 8th hr) the absorbancy of thecontrol culture lagged approximately 1 hr behindthat of either the glucose-containing or theglycerol-containing culture, differences in enzymeactivity did not appear to be related to differencesin growth of the cultures. The tryptophanase ofE. coli is known to be repressible by glucose(2, 6).Another manifestation of catabolite repression

is evident in Fig. 3. B. alvei excretes indole attwo stages in its growth cycle (8). "Early" indoleis excreted as a result of indole-3-glycerol phos-phate fission and is then reutilized in the trypto-

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FIG. 2. Effects of glucose anid glycerol oni trypto-phanase synthesis. Growth anid assay conditions wereas described in Materials and Methods. Cultures weresampled hourly and the growth was measured. Sampleswere centrifuged, and the supernatant fluids were re-tained for indole determination (see Fig. 3) and pyru-vate determination (see Fig. 5). The cells were washed,resuspended, toluene-treated, and assayed for trypto-phanase activity (above figure) anid L-serine dehydra-tase activity (see Fig. 4).

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FIG. 3. Effiects of glucose an1d glycerol onl excre-tion of indole durin1g growth. Conditions are as de-scribed in2 Fig. 2. Samples (0.S ml) of culture super-natant fluids were assayed for indole by the addition of3 ml of color reagent, incubation at room temperaturefor 20 mini, and measuremen1t of color in?tentsities atS68 nm.

phan synthetase-catalyzed biosynthesis of trypto-phan. The excretion of indole late in the growthcycle is a consequence of tryptophanase activity."Late" indole excretion was completely elimi-nated when the organisms were grown in thepresence of 1% concentrations of either glucoseor glycerol.

In all three cases, "early" indole was excretedand then reutilized, suggesting that tryptophansynthetase is probably not repressible by eitherglucose or glycerol. This possibility was sub-stantiated in C. W. Roth's studies on tryptophansynthetase in B. alvei (Ph.D. Thesis, Univ. ofIllinois, Urbana, 1969). The 1% glucose presentin media used in his experiments did not apprecia-bly affect the production and reutilization of

"early" indole or the level of tryptophan syn-thetase in the organisms. Furthermore, Freund-lich and Lichstein (2) have found not only thatglucose fails to repress tryptophan synthetasein E. coli, but also that high glucose levels resultin an increased enzyme level.The effect of glucose on L-serine dehydratase

synthesis differed markedly from the effect ofthe same compound on the synthesis of tryp-tophanase. The specific activity curves of thecontrol culture and the glucose-grown cultureare quite different in shape (Fig. 4). However,the areas under the curves are similar, indicatingthat the total amounts of enzyme formed duringthe growth cycle of the organism with and withoutglucose were the same. Again, differences inenzyme activity did not appear to be related todifferences in growth of the cultures (4th to 8thhr). The mechanism responsible for the displace-ment of L-serine dehydratase synthesis by glucoseis not clear at the present time. It may be notedthat glycerol exerted a repressive effect on thisenzyme also.

Pyruvate excretion in the absence of addedglucose or glycerol and in the presence of glucoseshowed a slight increase early in the exponentialgrowth phase and then dropped to a relativelylow constant level (Fig. 5). In the case of glycerol,however, pyruvate was excreted in copiousamounts during exponential growth. The excretedpyruvate is probably derived from glycerol.From the repression of the dehydratase byglycerol and the excretion of pyruvate, it may besuggested that the conversion of glycerol topyruvate is largely unregulated in comparison tothe conversion of glucose to pyruvate.

Optimal pH for enzyme activity. It has beenconcluded by Hoch et al. (9) that tryptophanasein crude extracts of B. alvei possesses maximalactivity at pH 9.0 in glycine-phosphate buffer,but that this optimum shifts to pH 7.5 to 8.4 inphosphate-acetate-borate buffer upon purifica-

FIG. 4. Efects of glucose alnd glycerol Onl L-seriunedehydratase syntthesis. Coniditionis are as described inFig. 2.

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FIG. 5. Effects of glucose aiid glycerol Oii excretionioJ pyruvate duriiig growth. Conditioiis are as describedin Fig. 2. Samples (0.25 ml) of conttrol aiid glucose-growiz culture superniatantfluids aiid 0. I-ml samples ofglycerol-grown culture supernataint fluids were assayedJor pyruvate as described in Materials aiid Methods.

tion of the enzyme. Analysis of additional experi-ments (Hoch, unpublished data) showed that thepurified enzyme has a pH optimum of 8.2 inpotassium phosphate buffer. The present investi-gation yielded a pH optimum of approximately8.2 for tryptophanase in toluene-treated wholecell preparations in potassium phosphate buffer(Fig. 6A).

L-Serine dehydratase activity in toluene-treatedwhole cell preparations in potassium phosphatebuffer was optimal at pH 7.5 (Fig. 6B).

Effect of monovalent cations and tris(hydroxy-methyl)aminomethane (Tris) on enzyme activity.Activation of tryptophanase by K+ and NH4+and inhibition of activity by Na+ have beendemonstrated by Hoch et al. (9). Furthermore,inhibition of enzyme activity in the presence ofTris has been suggested by Gopinathan andDeMoss (4, 5) to be the result of Schiff-baseformation between Tris and the coenzyme pyri-doxal phosphate, followed by dissociation ofthe enzyme into inactive subunits. The sameactivation by K+ and NH4+ and inhibition byNa+ and Tris were observed in the current in-vestigation (Table 4). All cations were presentin the form of phosphate buffer (pH 7.5) at afinal concentration of 0.09 M with respect tocation and 0.05 M with respect to phosphate. Tris,present at a final concentration of 0.05 M, wasadjusted to pH 7.5 with hydrochloric acid.

Examination of L-serine dehydratase activityin the presence of the same monovalent cationsyielded somewhat different results. All threecations stimulated enzyme activity (Table 4). Asin the case of tryptophanase, Tris was inhibitory

to dehydratase activity, though not to the samedegree. Whether the observed inhibition was aresult of partial dissociation of the enzyme intoinactive or partially active subunits has not beendetermined.

Affinity of tryptophanase and L-serine de-hydratase for their respective substrates. Thesaturation curve for the L-serine dehydratase-L-serine reaction (Fig. 7) and the double reciprocalplot of the data (Fig. 8) indicate that inhibitionof enzyme activity occurred at high substrateconcentrations. To test whether this inhibitionwas due to product accumulation, enzyme ac-tivity was examined in the presence of 0.1 ML-serine (the usual assay level) and increasing

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L-SERINE DEHYDRATASE AND TRYPTOPHANASE

concentrations of pyruvate. At initial pyruvatelevels ranging from 0 to 0.1 M, enzyme activityremained constant. Thus, the apparent substrateinhibition was actual inhibition.

In the case of tryptophanase, inhibition ofactivity also occurred at high tryptophan levelswhen the modified version of the assay procedureof Pardee and Prestidge was used. This methodprevents removal of indole as it is formed. Useof the modification of the assay procedure ofBoezi and DeMoss, whereby indole is removed

TABLE 4. Effect of monovalent cations and Trison tryptophanase and L-serine

dehydratase activities"

Cain Reac- Ez~ePer centEnzyme oraTrio tion Enzye of KcEnzyme or Tris final pH units activity

Tryptophanase K+ 7.63 0.0083 100NH4+ 7.45 0.0081 98Na+ 7.49 0.0023 28Tris 7.77 0.0015 18Water 8.22 0.0032 38

L-Serine dehy- K+ 7.59 0.272 100dratase NH4+ 7.53 0.289 106

Na+ 7.59 0.221 81Tris 7.71 0.127 47Water 7.72 0.174 64

a All cations were present in the form of phos-phate buffer (pH 7.5) at a final concentration of0.09 M with respect to cation and 0.05 M with re-spect to phosphate. Tris, at a final concentrationof 0.05 M, was adjusted to pH 7.5 with hydrochloricacid. The pH of cell suspensions in distilled waterwas 7.5, and the pH of the distilled water reactionmixtures was 7.3 at the onset of incubation.

FIG. 7. Affinities of tryptophanase for L-tryptophanand L-serine dehydratase for L-serine. Assay contditionswere as described in Materials and Methods, exceptthat various levels of the substrates were incorporatedinto the reaction mixtures.

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FIG. 8. Affinities of tryptophanase for L-tryptophanand L-serine dehydratase for L-serine. Double reciprocal(Lineweaver-Burk) plots of the data from Fig. 7.

by dissolution in the toluene overlay, eliminatedall traces of substrate inhibition at high trypto-phan concentrations.Note (Fig. 7) that the range of L-serine con-

centrations for maximal L-serine dehydrataseactivity is very narrow as compared to that ofL-tryptophan for maximal tryptophanase activity.The apparent Km (L-tryptophan) for trypto-

phanase, calculated from the data in Fig. 8, is0.267 mM. Km values of 0.272 mM and 0.273 mMfor L-tryptophan were obtained by Hoch et al.(9) and by S. R. O'Neil (Ph.D. Thesis, Univ. ofIllinois, Urbana, 1969), respectively, by usingpurified tryptophanase preparations. The valueobtained with toluene-treated whole cell prepara-tions is in good agreement with those obtainedfor the purified enzyme.The apparent Km (L-serine) for L-serine de-

hydratase, calculated from the data in Fig. 8,is 6.30 mm. O'Neil (Ph.D. Thesis, Univ. ofIllinois,Urbana, 1969) obtained a Km value of 0.129 Mfor the tryptophanase-catalyzed deamination ofL-serine. In view of the 20-fold difference inaffinities of the two enzymes for the same sub-strate and keeping in mind that the, concentrationof L-serine during enzyme assays was optimal(0.1 m) for the L-serine dehydratase-L-serinereaction, it seems reasonable to assume that thetotal L-serine deamination obtained in the currentinvestigation was a result of L-serine dehydrataseactivity and not of tryptophanase activity.

Specificity of L-serine dehydratase. Table 5shows the effect of L-serine dehydratase on variousamino acid substrates. L-Threonine and L-tryp-tophan were not cleaved in the presence of theenzyme. The efficiency of the enzyme in catalyzingthe degradation of D-serine was less than 1 %of that for the L-isomer. However, when subjected

819VOL. 101, 1970

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GRIFFITHS AND DEMOSS

TABLE 5. Effect of L-serine dehydratase 0ol amintoacid substrates

KeoPer centSubstrate Final acid of

concn formed L-serine

M ILmoleslmin

Expt AL-Serine 0.05 0.340 100D-Serine 0.10 0.000 0DL-a-Methylserlne 0.08 0.002 0.6DL-fl-Phenylserine 0.06 0.184 54DL-O-methylserine 0.08 0.000 0Cycloserine 0.10 0.000 0L-Threonine 0.08 0.004 1.2L-Tryptophan 0.02 0.000 0

Expt BL-Serine 0.10 0.456 100D-Serine 0.10 0.004 1L-Threonine 0.08 0.000 0

L-Serifne 0.10 0.214 47D-Serine 0.10

L-Serine 0.10 0 464 102L-Threonline 0.08

to equimolar concentrations of the two isomers,the enzyme was only one-half as effective incatalyzing the degradation of L-serine, suggestingthat D-serine is able to occupy an active site on'the enzyme. D-Serine has, in fact, been shownto be a competitive inhibitor of the L-serinedehydratase-L-serine reaction. In this capacity,D-serine exhibited a Ki of 4.56 mM (unpublisheddata).Of the serine analogues tested (Table 5), only

the j3-phenyl derivative was cleaved in the pres-ence of the enzyme and then only 54% as effec-tively as L-serine. Since the f3-phenylserine usedas the substrate was a racemic mixture, thereduced efficiency of the enzyme in catalyzingcleavage of the ,3-phenyl compound might beexplained as in the preceding paragraph; thatis, the D-isomer may serve as a competitiveinhibitor of the reaction.We conclude that the two enzymes in B. alvei

which catalyze the deamination of L-serine are

quite dissimilar in the physiological properties

investigated. The dehydratase is currently beingpurified in our laboratory, so that a more ac-

curate physical comparison of the two enzymes

can be achieved.

ACKNOWLEDGMENT

S. K. Griffiths was a predoctoral fellow-trainee supported byPublic Health Service microbiology training grant GM-S10 fromthe National Institute of General Medical Sciences. This investi-gation was supported by Public Health Service research grantsAI-2971 from the National Institute of Allergy and InfectiousDiseases and AM-11696 from the National Institute of Arthritisand Metabolic Diseases.

LITERATURE CITED1. Boezi, J. A., and R. D. DeMoss. 1961. Properties of a trypto-

phan transport system in Escherichia coli. Biochim. Bio-phys. Acta 49:471-484.

2. Freundlich, M., and H. C. Lichstein. 1962. Tryptophanase-tryptophan synthetase systems in Escherichia coll. II.Effect of glucose. J. Bacteriol. 84:988-995.

3. Friedemann, T. E., and G. E. Haugen. 1943. Pyruvic acid.IJ. The determination of keto acids in blood and urine.J. Biol. Chem. 147:415-442.

4. Gopinathan, K. P., and R. D. DeMoss. 1966. Reversibledissociation of tryptophanase into protein subunits. Proc.Nat. Acad. Sci. U.S.A. 56:1223-1225.

5. Gopinathan, K. P., and R. D. DeMoss. 1968. The role oftris(hydroxymethyl)aminomethane and cysteine in thedissociation of tryptophanase. Biochemistry 7:1685-1691.

6. Happold, F. C. 1950. Tryptophanase-tryptophan reaction.Advan. Enzymol. 10: 51-82.

7. Happold, F. C., and L. Hoyle. 1935. The coli-tryptophan-indole reaction. I. Enzyme preparations and their actionon tryptophan and some indole derivatives. Biochem. J.29:1918-1926.

8. Hoch, J. A., and R. D. DeMoss. 1965. Physiological effectsof a constitutive tryptophanase in Bacillus alvei. J. Bacteriol.90:604-610.

9. Hoch, J. A., F. J. Simpson, and R. D. DeMoss. 1966. Purifica-tion and some properties of tryptophanase from Bacillusalvei. Biochemistry 5:2229-2237.

10. Kay, W. W., and A. F. Gronlund. 1969. Amino acid trans-port in Pseudomonas aeruginosa. J. Bacteriol. 97:273-281.

11. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Ran-dall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

12. Newton, W. A., and E. E. Snell. 1962. An inducible trypto-phan synthetase in tryptophan auxotrophs of Escherichiacoli. Biochemistry 48:1431-1439.

13. Newton, W. A., and E. E. Snell. 1964. Catalytic properties oftryptophanase, a multifunctional pyridoxal phosphateenzyme. Proc. Nat. Acad. Sci. U.S.A. 51:382-389.

14. Pardee, A. B., and L. S. Prestidge. 1955. Induced formation ofserine and threonine deaminases by Escherichia coil. J.Bacteriol. 70:667-674.

15. Pardee, A. B., and L. S. Prestidge. 1961. The initial kinetics ofenzyme induction. Biochim. Biophys. Acta 49:77-88.

16. Vogel, H. J., and D. M. Bonner. 1956. Acetyl-ornithinase ofEscherichia coli: partial purification and properties. J.Biol. Chem. 218:97-106.

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